技术资料

The B cell receptor (BCR) signals together with a multi-component co-receptor complex to initiate B cell activation in response to antigen binding. Here,we take advantage of peroxidase-catalyzed proximity labeling combined with quantitative mass spectrometry to track co-receptor signaling dynamics in Raji cells from 10 s to 2 h after BCR stimulation. This approach enables tracking of 2,814 proximity-labeled proteins and 1,394 phosphosites and provides an unbiased and quantitative molecular map of proteins recruited to the vicinity of CD19,the signaling subunit of the co-receptor complex. We detail the recruitment kinetics of signaling effectors to CD19 and identify previously uncharacterized mediators of B cell activation. We show that the glutamate transporter SLC1A1 is responsible for mediating rapid metabolic reprogramming and for maintaining redox homeostasis during B cell activation. This study provides a comprehensive map of BCR signaling and a rich resource for uncovering the complex signaling networks that regulate activation. View Publication

Although platinum-based chemotherapy is the frontline regimen for colorectal cancer (CRC),drug resistance remains a major challenge affecting its therapeutic efficiency. However,there is limited research on the correlation between chemotherapy resistance and lipid metabolism,including PIK3CA mutant tumors. In this present study,we found that PIK3CA-E545K mutation attenuated cell apoptosis and increased the cell viability of CRC with L-OHP treatment in vitro and in vivo . Mechanistically,PIK3CA-E545K mutation promoted the nuclear accumulation of SREBP1,which promoted the transcription of Apolipoprotein A5 (APOA5). APOA5 activated the PPARγ signaling pathway to alleviate reactive oxygen species (ROS) production following L-OHP treatment,which contributed to cell survival of CRC cells. Moreover,APOA5 overexpression enhanced the stemness-related traits of CRC cells. Increased APOA5 expression was associated with PIK3CA mutation in tumor specimens and poor response to first-line chemotherapy,which was an independent detrimental factor for chemotherapy sensitivity in CRC patients. Taken together,this study indicated that PIK3CA-E545K mutation promoted L-OHP resistance by upregulating APOA5 transcription in CRC,which could be a potent target for improving L-OHP chemotherapeutic efficiency. Our study shed light to improve chemotherapy sensitivity through nutrient management in CRC. View Publication

Chimeric antigen receptor (CAR) T cell therapy has emerged as a powerful therapeutic approach against a range of hematologic malignancies. While the incorporation of CD28 or 4-1BB costimulatory signaling domains into CARs revolutionized immune responses,there is an exciting prospect of further enhancing CAR functionality. Here,we investigated the design of CD19 CARs enriched with distinct Toll-like receptor 4 (TLR4),myeloid differentiation primary response 88 (MyD88),or Toll/IL-1 domain-containing adaptor-inducing interferon (IFN)-β (TRIF) costimulatory domains. Screening of various designs identified several candidates with no tonic activity but with increased CD19 target cell-dependent interleukin (IL)-2 production. Human T cells transduced with the selected CAR construct exhibited augmented hIL-2 and hIFN-γ induction and cytotoxicity when cocultured with CD19-positive lymphoma and solid-tumor cell lines. RNA sequencing (RNA-seq) analysis demonstrated the upregulation of some genes involved in the innate immune response and T cell activation and proliferation. In experiments on a xenogeneic solid-tumor mice model,MyD88 and TLR4 CAR T cells exhibited prolonged remission. This study demonstrates that the integration of a truncated TLR4 signaling costimulatory domain could provide immunotherapeutic potential against both hematologic malignancies and solid tumors. View Publication

The processes that govern human haematopoietic stem cell (HSC) self-renewal and engraftment are poorly understood and challenging to recapitulate in culture to reliably expand functional HSCs 1 – 3 . Here we identify MYC target 1 (MYCT1; also known as MTLC) as a crucial human HSC regulator that moderates endocytosis and environmental sensing in HSCs. MYCT1 is selectively expressed in undifferentiated human haematopoietic stem and progenitor cells (HSPCs) and endothelial cells but becomes markedly downregulated during HSC culture. Lentivirus-mediated knockdown of MYCT1 prevented human fetal liver and cord blood (CB) HSPC expansion and engraftment. By contrast,restoring MYCT1 expression improved the expansion and engraftment of cultured CB HSPCs. Single-cell RNA sequencing of human CB HSPCs in which MYCT1 was knocked down or overexpressed revealed that MYCT1 governs important regulatory programmes and cellular properties essential for HSC stemness,such as ETS factor expression and low mitochondrial activity. MYCT1 is localized in the endosomal membrane in HSPCs and interacts with vesicle trafficking regulators and signalling machinery. MYCT1 loss in HSPCs led to excessive endocytosis and hyperactive signalling responses,whereas restoring MYCT1 expression balanced culture-induced endocytosis and dysregulated signalling. Moreover,sorting cultured CB HSPCs on the basis of lowest endocytosis rate identified HSPCs with preserved MYCT1 expression and MYCT1-regulated HSC stemness programmes. Our work identifies MYCT1-moderated endocytosis and environmental sensing as essential regulatory mechanisms required to preserve human HSC stemness. Our data also pinpoint silencing of MYCT1 as a cell-culture-induced vulnerability that compromises human HSC expansion. Subject terms: Haematopoietic stem cells,Self-renewal,Stem-cell niche,Endocytosis,Growth factor signalling View Publication

Acute myeloid leukemia (AML) is fatal in the majority of adults. Identification of new therapeutic targets and their pharmacologic modulators are needed to improve outcomes. Previous studies had shown that immunization of rabbits with normal peripheral WBCs that had been incubated with fluorodinitrobenzene elicited high titer antibodies that bound to a spectrum of human leukemias. We report that proteomic analyses of immunoaffinity-purified lysates of primary AML cells showed enrichment of scaffolding protein IQGAP1. Immunohistochemistry and gene-expression analyses confirmed IQGAP1 mRNA overexpression in various cytogenetic subtypes of primary human AML compared to normal hematopoietic cells. shRNA knockdown of IQGAP1 blocked proliferation and clonogenicity of human leukemia cell-lines. To develop small molecules targeting IQGAP1 we performed in-silico screening of 212,966 compounds,selected 4 hits targeting the IQGAP1-GRD domain,and conducted SAR of the ‘fittest hit’ to identify UR778Br,a prototypical agent targeting IQGAP1. UR778Br inhibited proliferation,induced apoptosis,resulted in G2/M arrest,and inhibited colony formation by leukemia cell-lines and primary-AML while sparing normal marrow cells. UR778Br exhibited favorable ADME/T profiles and drug-likeness to treat AML. In summary, AML shows response to IQGAP1 inhibition,and UR778Br,identified through in-silico studies,selectively targeted AML cells while sparing normal marrow. Subject terms: Cancer,Cell biology,Drug discovery,Immunology,Oncology View Publication

Adult T-cell leukemia/lymphoma (ATL) occurs after human T-cell leukemia virus type-1 (HTLV-1) infection with a long latency period exceeding several decades. This implies the presence of immune evasion mechanisms for HTLV-1-infected T cells. Although ATL cells have a CD4 + CD25 + phenotype similar to that of regulatory T cells (Tregs),they do not always possess the immunosuppressive functions of Tregs. Factors that impart effective immunosuppressive functions to HTLV-1-infected cells may exist. A previous study identified a new CD13 + Treg subpopulation with enhanced immunosuppressive activity. We,herein,describe the paired CD13 − (designated as MT-50.1) and CD13 + (MT-50.4) HTLV-1-infected T-cell lines with Treg-like phenotype,derived from the peripheral blood of a single patient with lymphoma-type ATL. The cell lines were found to be derived from HTLV-1-infected non-leukemic cells. MT-50.4 cells secreted higher levels of immunosuppressive cytokines,IL-10 and TGF-β,expressed higher levels of Foxp3,and showed stronger suppression of CD4 + CD25 − T cell proliferation than MT-50.1 cells. Furthermore,the CD13 inhibitor bestatin significantly attenuated MT-50.4 cell growth,while it did not for MT-50.1 cells. These findings suggest that CD13 expression may be involved in the increased Treg-like activity of MT-50.4 cells. Hence,MT-50.4 cells will be useful for in-depth studies of CD13 + Foxp3 + HTLV-1-infected cells. Subject terms: Cancer,Microbiology,Oncology View Publication

Dynamic regulation of gene expression is fundamental for cellular adaptation to exogenous stressors. P-TEFb-mediated pause-release of RNA polymerase II (Pol II) is a conserved regulatory mechanism for synchronous transcriptional induction in response to heat shock,but this pro-survival role has not been examined in the applied context of cancer therapy. Using model systems of pediatric high-grade glioma,we show that rapid genome-wide reorganization of active chromatin facilitates P-TEFb-mediated nascent transcriptional induction within hours of exposure to therapeutic ionizing radiation. Concurrent inhibition of P-TEFb disrupts this chromatin reorganization and blunts transcriptional induction,abrogating key adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent,synergistic therapeutic potential agnostic of glioma subtype,leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival. These studies reveal a central role for P-TEFb underpinning the early adaptive response to radiotherapy,opening avenues for combinatorial treatment in these lethal malignancies. Subject terms: CNS cancer,Paediatric cancer,Radiotherapy View Publication

Malaria parasite invasion to host erythrocytes is mediated by multiple interactions between merozoite ligands and erythrocyte receptors that contribute toward the development of disease pathology. Here,we report a novel antigen Plasmodium prohibitin “ Pf PHB2” and identify its cognate partner “Hsp70A1A” in host erythrocyte that plays a crucial role in mediating host-parasite interaction during merozoite invasion. Using small interfering RNA (siRNA)- and glucosamine-6-phosphate riboswitch (glmS) ribozyme-mediated approach,we show that loss of Hsp70A1A in red blood cells (RBCs) or Pf PHB2 in infected red blood cells (iRBCs),respectively,inhibit Pf PHB2-Hsp70A1A interaction leading to invasion inhibition. Antibodies targeting Pf PHB2 and monoclonal antibody therapeutics against Hsp70A1A efficiently block parasite invasion. Recombinant Pf PHB2 binds to RBCs which is inhibited by anti- Pf PHB2 antibody and monoclonal antibody against Hsp70A1A. The validation of Pf PHB2 to serve as antigen is further supported by detection of anti- Pf PHB2 antibody in patient sera. Overall,this study proposes Pf PHB2 as vaccine candidate and highlights the use of monoclonal antibody therapeutics for future malaria treatment. Subject areas: biochemistry,molecular biology,immunology View Publication

Cohesin plays a crucial role in the organization of topologically-associated domains (TADs),which influence gene expression and DNA replication timing. Whether epigenetic regulators may affect TADs via cohesin to mediate DNA replication remains elusive. Here,we discover that the histone demethylase PHF2 associates with RAD21,a core subunit of cohesin,to regulate DNA replication in mouse neural stem cells (NSC). PHF2 loss impairs DNA replication due to the activation of dormant replication origins in NSC. Notably,the PHF2/RAD21 co-bound genomic regions are characterized by CTCF enrichment and epigenomic features that resemble efficient,active replication origins,and can act as boundaries to separate adjacent domains. Accordingly,PHF2 loss weakens TADs and chromatin loops at the co-bound loci due to reduced RAD21 occupancy. The observed topological and DNA replication defects in PHF2 KO NSC support a cohesin-dependent mechanism. Furthermore,we demonstrate that the PHF2/RAD21 complex exerts little effect on gene regulation,and that PHF2’s histone-demethylase activity is dispensable for normal DNA replication and proliferation of NSC. We propose that PHF2 may serve as a topological accessory to cohesin for cohesin localization to TADs and chromatin loops,where cohesin represses dormant replication origins directly or indirectly,to sustain DNA replication in NSC. View Publication

Inflammatory pain results from the heightened sensitivity and reduced threshold of nociceptor sensory neurons due to exposure to inflammatory mediators. However,the cellular and transcriptional diversity of immune cell and sensory neuron types makes it challenging to decipher the immune mechanisms underlying pain. Here we used single-cell transcriptomics to determine the immune gene signatures associated with pain development in three skin inflammatory pain models in mice: zymosan injection,skin incision and ultraviolet burn. We found that macrophage and neutrophil recruitment closely mirrored the kinetics of pain development and identified cell-type-specific transcriptional programs associated with pain and its resolution. Using a comprehensive list of potential interactions mediated by receptors,ligands,ion channels and metabolites to generate injury-specific neuroimmune interactomes,we also uncovered that thrombospondin-1 upregulated by immune cells upon injury inhibited nociceptor sensitization. This study lays the groundwork for identifying the neuroimmune axes that modulate pain in diverse disease contexts. Subject terms: Neuroimmunology,Inflammation View Publication

Of all gynecologic cancers,epithelial-ovarian cancer (OCa) stands out with the highest mortality rates. Despite all efforts,90% of individuals who receive standard surgical and cytotoxic therapy experience disease recurrence. The precise mechanism by which leukemia inhibitory factor (LIF) and its receptor (LIFR) contribute to the progression of OCa remains unknown. Analysis of cancer databases revealed that elevated expression of LIF or LIFR was associated with poor progression-free survival of OCa patients and a predictor of poor response to chemotherapy. Using multiple primary and established OCa cell lines or tissues that represent five subtypes of epithelial-OCa,we demonstrated that LIF/LIFR autocrine signaling is active in OCa. Moreover,treatment with LIFR inhibitor,EC359 significantly reduced OCa cell viability and cell survival with an IC 50 ranging from 5-50 nM. Furthermore,EC359 diminished the stemness of OCa cells. Mechanistic studies using RNA-seq and rescue experiments unveiled that EC359 primarily induced ferroptosis by suppressing the glutathione antioxidant defense system. Using multiple in vitro,ex vivo and in vivo models including cell-based xenografts,patient-derived explants,organoids,and xenograft tumors,we demonstrated that EC359 dramatically reduced the growth and progression of OCa. Additionally,EC359 therapy considerably improved tumor immunogenicity by robust CD45 + leukocyte tumor infiltration and polarizing tumor-associated macrophages (TAMs) toward M1 phenotype while showing no impact on normal T-,B-,and other immune cells. Collectively,our findings indicate that the LIF/LIFR autocrine loop plays an essential role in OCa progression and that EC359 could be a promising therapeutic agent for OCa. Subject terms: Molecular medicine,Ovarian cancer View Publication

Establishing robust models of human myelinating Schwann cells is critical for studying peripheral nerve injury and disease. Stem cell differentiation has emerged as a key human cell model and disease motivating development of Schwann cell differentiation protocols. Human embryonic stem cells (hESCs) are considered the ideal pluripotent cell but ethical concerns regarding their use have propelled the popularity of human induced pluripotent stem cells (hiPSCs). Given that the equivalence of hESCs and hiPSCs remains controversial,we sought to compare the molecular and functional equivalence of hESC- and hiPSC-derived Schwann cells generated with our previously reported protocol. We identified only modest transcriptome differences by RNA sequencing and insignificant proteome differences by antibody array. Additionally,both cell types comparably improved nerve regeneration and function in a chronic denervation and regeneration animal model. Our findings demonstrate that Schwann cells derived from hESCs and hiPSCs with our protocol are molecularly comparable and functionally equivalent. Subject areas: Neuroscience,Cell biology,Stem cells research,Transcriptomics View Publication